DNA virus microRNA and methods for inhibiting same

ABSTRACT

The invention relates to isolated nucleic acid molecules comprising the sequence of a human cytomegalovirus microRNA. In another embodiment, the invention relates to single stranded DNA virus microRNA molecules comprising the sequence of a human cytomegalovirus microRNA. The invention also relates to the anti-DNA virus microRNA molecules.

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/925,363 filed on Aug. 24, 2004, which is acontinuation-in-part of U.S. patent application Ser. No. 10/819,098filed on Apr. 5, 2004. The specifications of U.S. patent applicationSer. Nos. 10/925,363 and 10/819,098 are hereby incorporated by referencein their entirety.

This invention described in this application was made with funds fromthe National Institutes of Health, Grant Number R01-GM068476-01. TheUnited States Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

MicroRNAs are small RNA molecules of about 22 nucleotides. ThesemicroRNA molecules can control gene expression in a sequence specificmanner in a wide variety of organisms.

In many organisms, RNA silencing mediated by double-stranded RNA(dsRNA), such as siRNA and microRNA, is part of an innate immuneresponse against RNA viruses and transposable elements. Counter defensestrategies to thwart the host response were found in, for example, plantviruses and the insect Flock House virus. These viruses expressinhibitors, e.g., dsRNA-binding proteins, that interfere with the hostcell RNA silencing machinery.

For example, microRNAs are reported to block translation after partiallyhybridizing to the non-coding 3′ region of mRNAs of target genes. Thegenes targeted by microRNAs largely remain to be characterized. However,there is growing evidence that microRNAs are implicated in variousdiseases and illnesses. For instance, drosophila microRNAs have beenshown to target genes involved in apoptosis, and B-cell chroniclymphocytic leukemia has been linked to the deletion of two microRNAs.

However, to date, the existence of microRNA encoded by mammalian viruseshave not been reported. Identifying mammalian virus microRNAs, and, ifthey exist, understanding their biological function would facilitate thedevelopment of new anti-viral drugs.

Therefore, there is a need to identify viral microRNAs, and for newmaterials and methods that can help elucidate the function of known andfuture virus microRNAs.

Due to the ability of microRNAs to induce RNA degradation or represstranslation of mRNA which encode important proteins, there is also aneed for novel molecules that inhibit DNA virus microRNA-inducedcleavage or translation repression of target mRNAs.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to an isolated nucleic acidmolecule comprising the sequence of a DNA virus microRNA.

In another embodiment, the invention relates to an isolated singlestranded DNA virus microRNA molecule. The molecule comprises a minimumof ten moieties and a maximum of fifty moieties on a molecular backbone,the molecular backbone comprising backbone units. Each moiety comprisesa base bonded to a backbone unit wherein at least ten contiguous baseshave the same sequence as a sequence of bases in a DNA virus microRNAmolecule, except that up to thirty percent of the bases pairs may bewobble base pairs, and up to 10% of the contiguous bases are additions,deletions, mismatches, or combinations thereof, and no more than fiftypercent of the contiguous moieties contain deoxyribonuleotide backboneunits.

In a further embodiment, the invention relates to an isolated singlestranded anti-DNA virus microRNA molecule. The anti-DNA virus microRNAmolecule comprises a minimum of ten moieties and a maximum of fiftymoieties on a molecular backbone, the molecular backbone comprisingbackbone units. Each moiety comprising a base bonded to a backbone unit,each base forming a Watson-Crick base pair with a complementary basewherein at least ten contiguous bases have a sequence complementary to acontiguous sequence of bases in a DNA virus microRNA molecule, exceptthat up to thirty percent of the bases pairs may be wobble base pairs,and up to 10% of the contiguous bases are additions, deletions,mismatches, or combinations thereof; no more than fifty percent of thecontiguous moieties contain deoxyribonuleotide backbone units; and themolecule is capable of inhibiting microRNP activity.

In yet a further embodiment, the invention relates to a method forinhibiting microRNP activity in a cell. The microRNP comprises a DNAvirus microRNA molecule, the DNA virus microRNA molecule comprising asequences of bases complementary to the sequence of bases in a singlestranded anti-DNA virus microRNA molecule. The method comprisesintroducing into the cell a single-stranded anti-DNA virus microRNAmolecule comprising a sequence of a minimum of ten moieties and amaximum of fifty moieties on a molecular backbone, the molecularbackbone comprising backbone units, each moiety comprising a base bondedto a backbone unit, each base forming a Watson-Crick base pair with acomplementary base, wherein at least ten contiguous bases of theanti-DNA virus microRNA molecule are complementary to the DNA virusmicroRNA, except that up to thirty percent of the bases may besubstituted by wobble base pairs, and up to ten percent of the at leastten moieties are addition, deletions, mismatches, or combinationsthereof, and no more than fifty percent of the contiguous moietiescontain deoxyribonuleotide backbone units.

In yet another embodiment, the invention relates to a method fortreating a DNA virus infection in a mammal in need thereof. The methodcomprises introducing into the mammal an anti-DNA virus microRNAmolecule.

In another embodiment, the invention relates to an isolated microRNPcomprising an isolated nucleic acid molecule described herein.

In a further embodiment, the invention relates to an isolated microRNPcomprising an isolated single stranded DNA virus microRNA molecule.

In yet a further embodiment, the invention relates to an isolatednucleic acid sequence comprising any one of the sequence of a DNA virusmicroRNA shown in Tables A1 or A2.

In yet another embodiment, the invention relates to an isolated singlestranded DNA virus microRNA molecule comprising a minimum of tenmoieties and a maximum of fifty moieties on a molecular backbone. Themolecular backbone comprising backbone units, each moiety comprising abase bonded to a backbone unit wherein: at least ten contiguous baseshave the same sequence as any one of the sequence of bases in a DNAvirus microRNA molecule shown in Tables A1 or A2, except that up tothirty percent of the bases pairs may be wobble base pairs, and up to10% of the contiguous bases are additions, deletions, mismatches, orcombinations thereof; and no more than fifty percent of the contiguousmoieties contain deoxyribonuleotide backbone units.

In another embodiment, the invention relates to an isolated singlestranded anti-DNA virus microRNA molecule comprising a minimum of tenmoieties and a maximum of fifty moieties on a molecular backbone. Themolecular backbone comprising backbone units, each moiety comprising abase bonded to a backbone unit, each base forming a Watson-Crick basepair with a complementary base wherein at least ten contiguous baseshave a sequence complementary to a contiguous sequence of bases in thesequence of bases in any one of the DNA virus microRNA molecule shown inTables A1 or A2, except that up to thirty percent of the bases pairs maybe wobble base pairs, and up to 10% of the contiguous bases areadditions, deletions, mismatches, or combinations thereof; no more thanfifty percent of the contiguous moieties contain deoxyribonuleotidebackbone units; and the molecule is capable of inhibiting microRNPactivity.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the modified nucleotide units discussed in thespecification. B denotes any one of the following nucleic acid bases:adenosine, cytidine, guanosine, thymine, or uridine.

FIG. 2. EBV expresses microRNAs. (A) Diagram of the microRNA containingsegments of the EBV genome. Latent genes are indicated with white boxes,lytic genes with black boxes, previously known non-coding RNAs with blueand newly identified microRNAs with red. Promoters active at latentstages (I, II, or III) are illustrated as white pennants, those activeat lytic stage as black pennants, and those active at all stages as graypennants. The intronic segments within the BARTs region are indicated asdashed lines, the exonic segments with bold bars. (B) Predictedfold-back precursors of the EBV microRNAs. The mature microRNA ishighlighted in red. An asterisk is used to denote a low abundant smallRNA that was cloned from the strand opposite to the microRNA-BHRF1-2strand. (C) Northern blots for EBV microRNAs using total RNA isolatedfrom uninfected BL-41 (−) and EBV-infected BL41/95 (+) cells. Theexpression of human miR-16 (Table S1) was also examined for reference.The position of migration of the mature microRNAs (miR) and itsfold-back precursors (miR-L) are indicated. Equal loading of the gelbefore transfer to the membrane was monitored by ethidium bromidestaining of the tRNA band. (D) Northern blots for EBV microRNAs usingtotal RNA isolated from various Hodgkin and Burkitt lymphoma cell lines.The latency stage for EBV positive lines is indicated in parentheses.The numbers below the miR signals indicate relative signal intensitywith respect to BL41/95 signals after normalizing for gel loading usingthe U6 snRNA signal.

FIG. 3. Schematic representation of miR-BART2-guided cleavage of BALF5mRNA. Lytic genes are shown as black boxes and genes for which theexpression has not been characterized are indicated in gray (GenBankentry V01555). The miR-BART2 sequence is aligned relative to thenucleotide sequence and the processing site of the BALF5 mRNA. Theprediction position of BALF5 mRNA cleavage coincides with the mappedterminus of the 3.7 kb processed form.

FIG. 4. Genomic positions and foldback structures of KSHV mRNAs. (A)Genomic positions of KSHV microRNAs. Solid arrows indicate open readingframes (ORF) conserved in Herpes Saimiri virus, open arrows indicate theunique KSHV ORFs. Repeat regions are shown as small filled rectanglesabove the ORFs. Cloned mRNAs are shown as dotted lines. The two possiblepromoters for K12 transcript are indicated as a black arrow, and K12transcripts as a black lines, the intronic region in the largertranscript is depicted as a break in the line. The thick grey arrowsshow ORF for Kaposin proteins A, B and C. (B) Foldback precursors ofKSHV microRNAs. The cloned mature microRNAs are highlighted in red.

FIG. 5. KSHV mRNAs are differentially regulated upon induction of thelytic cycle. Northern blots for KSHV miR-K1a, miR-K6 and miR-K7 madefrom total RNA isolated from a KSHV negative (BJAB) cell line and fromBCBL1 cells at 24 h, 48 h and 72 h after TPA treatment.

FIG. 6. Genomic positions and secondary structures of HCMV mRNAs. (A)Diagram of the mRNA-containing fragments of the HCMV genome. Terminalrepeats are shown as grey boxes and triangles. Cloned mRNAs are shown asdotted red lines. The mRNAs encoded from the (+)-strand of the genomeare shown above the genome, those deriving from the (−)-strand of thegenome are shown below. The arrows indicate the orientation of the viralORF's. (B) Predicated foldback precursors of HCMV microRNAs. The clonedmature microRNAs are highlighted in red. An asterisk is used tohighlight low-abundance small RNA that was cloned from the strandopposite from miR-UL1 strand.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered DNA virus-encoded microRNAs. Thus, in oneembodiment, the invention relates to an isolated single stranded DNAvirus microRNA molecule.

MicroRNA molecules are known in the art (see, for example, Bartel, Cell,2004, 116, 281-297 for a review on microRNA molecules). The article byBartel is hereby incorporated by reference. Such molecules are derivedfrom genomic loci and are produced from specific microRNA genes.

Mature microRNA molecules are processed from precursor transcripts thatform local hairpin structures. The hairpin structures are typicallycleaved by an enzyme known as Dicer, generating thereby one microRNAduplex. See the above reference by Bartel.

Usually, one of the two strands of a microRNA duplex is packaged in amicroRNA ribonucleoprotein complex (microRNP). A microRNP in, forexample, humans, also includes the proteins eIF2C2, helicase, e.g,Gemin3, and Gemin 4.

Unmodified DNA Virus microRNA Molecules

In one embodiment, the invention relates to an isolated nucleic acidmolecule comprising a DNA virus microRNA sequence or a DNA virus hairpinprecursor sequence. In addition to the sequence of the DNA virusmicroRNA or hairpin precursor, the nucleic acid molecule may also haveone or more additional nucleotides. Any nucleotide can be added. Thereis no upper limit to the additional number of nucleotides. Typically, nomore than about 500 nucleotides, and preferably no more than about 300nucleotides are added to the DNA virus microRNA sequence or hairpinprecursor sequence. In one embodiment, the DNA virus microRNA is part ofa hairpin precursor sequence of fragment thereof.

The DNA virus microRNA can be inserted into a vector, such as, forexample, a recombinant vector. Typically, to construct such arecombinant vector containing a DNA virus microRNA, the hairpinprecursor sequence which contains the DNA virus microRNA sequence, isincorporated into the vector. See for example, Chen et al. Science 2004,303:83-86.

The recombinant vector may be any recombinant vector, such as a plasmid,a cosmid or a phage. Recombinant vectors generally have an origin ofreplication. The vector may be, for example, a viral vector, such as anadenovirus vector or an adeno-associated virus (AAV) vector. See forexample: Ledley 1996, Pharmaceutical Research 13:1595-1614 and Verma etal. Nature 1997, 387:239-242.

The vector may further include a selectable marker, such as for instancea drug resistance marker or a detectable gene marker, such asβ-galactosidase.

In a preferred embodiment, the nucleic acid molecule consists of a DNAvirus microRNA sequence or a hairpin precursor sequence. In anotherpreferred embodiment, the nucleic acid molecule consists of any one ofthe DNA virus microRNA sequence or hairpin precursor sequence shown inTable A, Table A1 or Table A2.

The DNA virus can be any DNA virus known to those skilled in the art.Preferably, the DNA virus infects mammalian cells. Examples of mammalsinclude laboratory animals, such as dogs and cats, farm animals, such ascows, horses and sheeps, laboratory animals, such as rats, mice andrabbits, and primates, such as monkeys and humans.

The DNA virus can be a single stranded or double stranded DNA virus.Examples of single stranded and double stranded DNA viruses are listedin Table B.

In one embodiment, the DNA virus is Epstein barr virus (EBV). Examplesof EBV microRNA's and the corresponding hairpin precursor sequences areshown in Table A.

In another embodiment, the DNA virus is Kaposi's sarcoma-associatedherpesvirus, also known as herpesvirus 8 (KSHV). Examples of KSHVmicroRNA's and the corresponding hairpin precursor sequences are shownin Table A1.

In yet another embodiment, the DNA virus is cytomegalovirus (HCMV).Examples of HCMV microRNA's and the corresponding hairpin precursorsequences are shown in Table A2.

The sequence of the isolated DNA virus microRNA molecules can be a DNAor RNA molecule. Sequences of nucleic acid molecules shown in Tables A,A1 and A2 are shown having uracil bases. Uracil bases occur inunmodified RNA molecules. The invention also includes unmodified DNAmolecules. The sequence of bases of the unmodified DNA molecule is thesame as the unmodified RNA molecules, except that in the unmodified DNAmolecule, the uracil bases are replaced with thymine bases. TABLE A EBVmicroRNA's and Hairpin Precursor Sequences microRNA Sequence HairpinPrecursor microRNA Sequence* Virus 5′→ 3′ (5′→ 3′) EBVUAACCUGAUCAGCCCCGGAGUU UAUUAACCUGAUCAGCCCCGGAGUU GCCUGUUUUCAUCACUAACCCCGGGCCUGAAGAGGUUGACAA UAUCUUUUGCGGCAGAAAUUGAACUUUUAAAUUCUGUUGCAGCAGAUAGCUGAUACCCAAU GUUAUCUUUUGCGGCAGAAAUUGAAAGUAACGGGAAGUGUGUAAGCACAC UCUAACGGGAAGUGUGUAAGCACACACGUAAUUUGCAAGCGGUGCUUCACGCUCUUCGUUAAAAU UCUUAGUGGAAGUGACGUGCUCGGGGUCUUAGUGGAAGUGACGUGCUGUGAAUACAG GUCCAUAGCACCGCUAUCCACUAUGUCUCGCCCGUAUUUUCUGCAUUCGCCCUUGC ACUAUUUUCUGCAUUCGCCCUUGCGUGUCCAUUGUUGCAAGGAGCGAUUUGGAGAAAAUAAA*In bold, mature microRNA sequence. In italics, a low abundant sequencecorresponding to the non-functional strand of the microRNA

TABLE A1 KSHV microRNA's and Hairpin Precursor Sequences microRNASequence Hairpin Precursor microRNA Sequence* Virus 5′→ 3′ (5′→ 3′) KSHVUAGUGUUGUCCCCCCGAGUGGC CUGGAGGCUUGGGGCGAUACCACCACUCGUUUGUCUGUUGGCGAUUAGUGUUGUCCCCCCGAGUGGCCAG UGGUGUUGUCCCCCCGAGUGGCCUGGAGGCUUGGGGCGAUACCACCACUCGUUUGUCUGUUG GCGAUUGGUGUUGUCCCCCCGAGUGGCCAGACCCAGCUGCGUAAACCCCGCU GGGUCUACCCAGCUGCGUAAACCCCGCUGCGUAAACACAGCUGGGUAUACGCAGCUGCGUAAACCC CUGGGUAUACGCAGCUGCGUAAGGUCUACCCAGCUGCGUAAACCCCGCUGCGUAAACACAGC UGGGUAUACGCAGCUGCGUAAACCCCCAGCAGCACCUAAUCCAUCGG CUUGUCCAGCAGCACCUAAUCCAUCGGCGGUCGGGCUGAUGGUUUUCGGGCUGUUGAGCGAG UGAUGGUUUUCGGGCUGUUGAGCUUGUCCAGCAGCACCUAAUCCAUCGGCGGUCGGGCUGAU GGUUUUCGGGCUGUUGAGCGAGUAGGAUGGCUGGAACUUGCCGG UGACCUAGGUAGUCCCUGGUGCCCUAAGGGUCUACAUCAAGCACUUAGGAUGCCUGGAACUUGCCGGUCA AGCUAAACCGCAGUACUCUAGGAUAACUAGCUAAACCGCAGUACUCUAGGGCAUUCAUUUG UUACAUAGAAUACUGAGGCCUAGCUGAUUAUUAGAAUACUGAGGCCUAGCUGA AUAACUAGCUAAACCGCAGUACUCUAGGGCAUUCAUUUGUUACAUAGAAUACUGAGGCCUAGCUGAUUAU UCACAUUCUGAGGACGGCAGCGGGCUAUCACAUUCUGAGGACGGCAGCGACGUGUGUCUAA CGUCAACGUCGCGGUCACAGAAUGUGACACCUCGCGGUCACAGAAUGUGACAC GGCUAUCACAUUCUGAGGACGGCAGCGACGUGUGUCUAAGGUCAACGUCGCGGUCACAGAAUGUGACACC AUUACAGGAAACUGGGUGUAAGGGAUUACAGGAAACUGGGUGUAAGCUGUACAUAAUCCCC GGCAGCACCUGUUUCCUGCAACCCUCGUUGAUCCCAUGUUGCUGGCGCUC GCGUUGAGCGCCACCGGACGGGGAUUUAUGCUGUAUCUUACUACCAUGAUCCCAUGUUGCUGGCGCUCACGG UUAAUGCUUAGCCUGUGUCCGACGCUUUGGUCACAGCUUAAACAUUUCUAGGGCGGUGUUAU GAUCCUUAAUGCUUAGCCUGUGUCCGAUGCGUAGGCGCGACUGAGAGAGCACG CGCGCACUCCCUCACUAACGCCCCGCUUUUGUCUGUUGGAAGCAGCUAGGCGCGACUGAGAGAGCACGCG*In bold, the mature microRNA sequence.

TABLE A2 HCMV microRNA's and Hairpin Precursor Sequences microRNASequence Hairpin Precursor microRNA Sequence* Virus 5′→ 3′ (5′→ 3′) HCMVUAACUAGCCUUCCCGUGAGAGU CCUGUCUAACUAGCCUUCCCGUGAGAGUUUAUGAACAUGUAUCUCACCAGAAUGCUAGUUUGUAGAGG UCACCAGAAUGCUAGUUUGUAGCCUGUCUAACUAGCCUUCCCGUGAGAGUUUAUGAACAUGU AUCUCACCAGAAUGCUAGUUUGUAGAGGUCGUUGAAGACACCUGGAAAGA CCACGUCGUUGAAGACACCUGGAAAGAGGACGUUCGGUCGGGCACGUUCUUUCCAGGUGUUUUCAACGUGCGUGG AAGUGACGGUGAGAUCCAGGCUGACAGCCUCCGGAUCACAUGGUUACUCAGCGUCUGCCAGC CUAAGUGACGGUGAGAUCCAGGCUGUCUCGUCCUCCCCUUCUUCACCGC AGCAGGUGAGGUUGGGGCGGACAACGUGUUGCGGAUUGUGGCGAGAACGUCGUCCUCCCCUUCUUCACCGCC UGACAAGCCUGACGAGAGCGUUUGAACGCUUUCGUCGUGUUUUUCAUGCAGCUUUUACAGAC CAUGACAAGCCUGACGAGAGCGUUCAUUAUGAUAGGUGUGACGAUGUC GGAGGCUUUCGCCACACCUAUCCUGAAAGCGUUGCAUUCUUUAUGAUAGGUGUGACGAUGUCUUU AACCGCUCAGUGGCUCGGACCGUGUGAACCGCUCAGUGGCUCGGACCGCCGGCUGUUUCUG CGCACCGGUCCGAACGCUAGGUCGGUUCUCAAGCGGUCUGUUCAGGUGGAUGA CGGUUAGCGGUCUGUUCAGGUGGAUGAGGGCUCUUCAGGGUCGGGCACUCGGCUGUGCCUGUCAUCCACUUGGAGAGCU CCCGCGGUCCGAUCCACUUGGAGAGCUCCCGCG CGGUUAGCGGUCUGUUCAGGUGGAUGAGGGCUCUUCACGGUCGGGCACUCGGCUGUGCCUGUCAUCCACUUGGAGAGCU CCCGCGGUCCGGAUUGUGCCCGGACCGUGGGCG CACGGUUGAUUGUGCCCGGACCGUGGGCGCGACGAAACCCACCGUCACGGUCCGAGCACAUCCAAACGUG*In bold, the mature microRNA sequence

TABLE B Single Stranded and Double Stranded DNA Viruses Family SubfamilyGenus Type species The dsDNA Viruses Poxviridae ChordopoxvirinaeOrthopoxvirus Vaccinia virus Parapoxvirus Orf virus LeporipoxvirusMyxoma virus Molluscipoxvirus Molluscum contagiosum virus HerpesviridaeAlphaherpesvirinae Simplexvirus Human herpesvirus 1 Varicellovirus Humanherpesvirus 3 Betaherpesvirinae Cytomegalovirus Human herpesvirus 5(HCMV) Muromegalovirus Murid herpesvirus 1 Roseolovirus Humanherpesvirus 6 Gammaherpesvirinae Lymphocryptovirus Human herpesvirus 4(EBV) Rhadinovirus Saimiriine herpesvirus 2 Rhadinovirus Humanherpesvirus 8 (KSHV) Adenoviridae Mastadenovirus Human adenovirus CPolyomaviridae Polyomavirus Simian virus 40 PapillomaviridaePapillomavirus Cottontail rabbit papillomavirus The ssDNA VirusesParvoviridae Parvovirinae Parvovirus Mice minute virus Erythrovirus B19virus Dependovirus Adeno-associated virus 2Analogs of DNA Virus microRNA Molecules

In another embodiment, the invention relates to analogs of DNA virusmicroRNAs or hairpin precursors described above, including those havingthe sequences shown in Table A, Table A1 or Table A2. In thisembodiment, the DNA virus microRNA molecule comprises a minimum numberof ten moieties, preferably a minimum of thirteen, more preferably aminimum of fifteen, even more preferably a minimum of eighteen, and mostpreferably a minimum of 21 moieties.

The DNA virus microRNA molecule comprises a maximum number of fiftymoieties, preferably a maximum of forty, more preferably a maximum ofthirty, even more preferably a maximum of twenty-five, and mostpreferably a maximum of twenty-three moieties. A suitable range ofminimum and maximum numbers of moieties may be obtained by combining anyof the above minima with any of the above maxima.

Each moiety comprises a base bonded to a backbone unit. In thisspecification, a base refers to any one of the nucleic acid basespresent in DNA or RNA. The base can be a purine or pyrimidine. Examplesof purine bases include adenine (A) and guanine (G). Examples ofpyrimidine bases include thymine (T), cytosine (C) and uracil (U). Eachbase of the moiety forms a Watson-Crick base pair with a complementarybase.

Watson-Crick base pairs as used herein refer to the hydrogen bondinginteraction between, for example, the following bases: adenine andthymine (A-T); adenine and uracil (A-U); and cytosine and guanine (C-G).The adenine can be replaced with 2,6-diaminopurine without compromisingbase-pairing.

The backbone unit may be any molecular unit that is able to stably bindto a base and to form an oligomeric chain. Suitable backbone units arewell known to those in the art.

For example, suitable backbone units include sugar-phosphate groups,such as the sugar-phosphate groups present in ribonucleotides,deoxyribonucleotides, phosphorothioate deoxyribose groups, N′3-N′5phosphoroamidate deoxyribose groups, 2′O-alkyl-ribose phosphate groups,2′-O-alkyl-alkoxy ribose phosphate groups, ribose phosphate groupcontaining a methylene bridge, 2′-fluororibose phosphate groups,morpholino phosphoroamidate groups, cyclohexene groups, tricyclophosphate groups, and amino acid molecules.

Preferably, the DNA virus microRNA molecule comprises at least onemoiety which confers increased nuclease resistance. Such moleculescomprise at least one moiety that is not recognized by a nuclease.Therefore, the nuclease resistance of the molecule is increased comparedto a sequence containing only unmodified ribonucleotide, unmodifieddeoxyribonucleotide or both. Such modified moieties are well known inthe art, and were reviewed, for example, by Kurreck, Eur. J. Biochem.270, 1628-1644 (2003).

The nuclease resisted can be an exonuclease, an endonuclease, or both.The exonuclease can be a 3′→5′ exonuclease or a 5′→3′ exonuclease.Examples of 3′→5′ human exonuclease include PNPT1, Werner syndromehelicase, RRP40, RRP41, RRP42, RRP45, and RRP46. Examples of 5′→3′exonuclease include XRN2, and FEN1. Examples of endonucleases includeDicer, Drosha, RNase4, Ribonuclease P, Ribonuclease H1, DHP1, ERCC-1 andOGG1. Examples of nucleases which function as both an exonuclease and anendonuclease include APE1 and EXO1.

A modified moiety can occur at any position in the DNA virus microRNAmolecule. For example, to protect the DNA virus microRNA moleculeagainst 3′→5′ exonucleases, the molecule can have at least one modifiedmoiety at the 3′ end of the molecule and preferably at least twomodified moieties at the 3′ end. If it is desirable to protect themolecule against 5′→3′ exonuclease, the DNA virus microRNA molecule canhave at least one modified moiety and preferably at least two modifiedmoieties at the 5′ end of the molecule. The DNA virus microRNA moleculecan also have at least one and preferably at least two modified moietiesbetween the 5′ and 3′ end of the molecule to increase resistance of themolecule to endonucleases. Preferably, at least about 10%, morepreferably at least about 25%, even more preferably at least about 50%,and further more preferably at least about 75%, and most preferablyabout 95% of the moieties are modified. In one embodiment, all of themoieties are nuclease resistant.

In another embodiment, the DNA virus microRNA molecule comprises atleast one modified deoxyribonucleotide moiety. Suitable modifieddeoxyribonucleotide moieties are known in the art.

A suitable example of a modified deoxyribonucleotide moiety is aphosphorothioate deoxyribonucleotide moiety. See structure 1 in FIG. 1.A DNA virus microRNA molecule comprising phosphorothioatedeoxyribonucleotide moieties is generally referred to asphosphorothioate (PS) DNA. See, for example, Eckstein, Antisense NucleicAcids Drug Dev. 10, 117-121 (2000).

Another suitable example of a modified deoxyribonucleotide moiety is anN′3-N′5 phosphoroamidate deoxyribonucleotide moiety. See structure 2 inFIG. 1. An oligonucleotide molecule comprising phosphoroamidatedeoxyribonucleotide moieties is generally referred to asphosphoroamidate (NP) DNA. See, for example, Gryaznov et al., J. Am.Chem. Soc. 116, 3143-3144 (1994).

In another embodiment, the molecule comprises at least one modifiedribonucleotide moiety. Suitable modified ribonucleotide moieties areknown in the art.

A suitable example of a modified ribonucleotide moiety is aribonucleotide moiety that is substituted at the 2′ position. Thesubstituents at the 2′ position may, for example, be a C₁ to C₄ alkylgroup. The C₁ to C₄ alkyl group may be saturated or unsaturated, andunbranched or branched. Some examples of C₁ to C₄ alkyl groups includeethyl, isopropyl, and allyl. The preferred C₁ to C₄ alkyl group ismethyl. See structure 3 in FIG. 1. An oligoribonucleotide moleculecomprising ribonucleotide moieties substituted at the 2′ position with aC₁ to C₄ alkyl group is generally referred to as a 2′-O-(C₁-C₄ alkyl)RNA, e.g., 2′-O-methyl RNA (OMe RNA).

Another suitable example of a substituent at the 2′ position of amodified ribonucleotide moiety is a C₁ to C₄ alkoxy -C₁ to C₄ alkylgroup. The C₁ to C₄ alkoxy (alkyloxy) and C₁ to C₄ alkyl group maycomprise any of the alkyl groups described above. The preferred C₁ to C₄alkoxy —C₁ to C₄ alkyl group is methoxyethyl. See structure 4 in FIG. 1.An oligonucleotide molecule comprising more than one ribonucleotidemoiety that is substituted at the 2′ position with a C₁ to C₄ alkoxy-C₁to C₄ alkyl group is referred to as a 2′-O—(C₁ to C₄ alkoxy —C₁ to C₄alkyl) RNA, e.g., 2′-O-methoxyethyl RNA (MOE RNA).

Another suitable example of a modified ribonucleotide moiety is aribonucleotide that has a methylene bridge between the 2′-oxygen atomand the 4′-carbon atom. See structure 5 in FIG. 1. Anoligoribonucleotide molecule comprising ribonucleotide moieties that hasa methylene bridge between the 2′-oxygen atom and the 4′-carbon atom isgenerally referred to as locked nucleic acid (LNA). See, for example,Kurreck et al., Nucleic Acids Res. 30, 1911-1918 (2002); Elayadi et al.,Curr. Opinion Invest. Drugs 2, 558-561 (2001); Ørum et al., Curr.Opinion Mol. Ther. 3, 239-243 (2001); Koshkin et al., Tetrahedron 54,3607-3630 (1998); Obika et al., Tetrahedron Lett. 39, 5401-5404 (1998).Locked nucleic acids are commercially available from Proligo (Paris,France and Boulder, Colo., USA).

Another suitable example of a modified ribonucleotide moiety is aribonucleotide that is substituted at the 2′ position with fluoro group.Such 2′-fluororibonucleotide moieties are known in the art. Moleculescomprising 2′-fluororibonucleotide moieties are generally referred toherein as 2′-fluororibo nucleic acids (FANA). See structure 7 in FIG. 1.Damha et al., J. Am. Chem. Soc. 120, 12976-12977 (1998).

In another embodiment, the DNA virus microRNA molecule comprises atleast one base bonded to an amino acid residue. Moieties that have atleast one base bonded to an amino acid residue will be referred toherein as peptide nucleic acid (PNA) moieties. Such moieties arenuclease resistance, and are known in the art. Molecules having PNAmoieties are generally referred to as peptide nucleic acids. Seestructure 6 in FIG. 1. Nielson, Methods Enzymol. 313, 156-164 (1999);Elayadi, et al, id.; Braasch et al., Biochemistry 41, 4503-4509 (2002),Nielsen et al., Science 254, 1497-1500 (1991).

The amino acids can be any amino acid, including natural or non-naturalamino acids. Naturally occurring amino acids include, for example, thetwenty most common amino acids normally found in proteins, i.e., alanine(Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine(Cys), glutamine (Glu), glutamic acid (Glu), glycine (Gly), histidine(His), isoleucine (Ileu), leucine (Leu), lysine (Lys), methionine (Met),phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr),tryptophan, (Trp), tyrosine (Tyr), and valine (Val).

The non-natural amino acids may, for example, comprise alkyl, aryl, oralkylaryl groups. Some examples of alkyl amino acids includeα-aminobutyric acid, β-aminobutyric acid, γ-aminobutyric acid,δ-aminovaleric acid, and ε-aminocaproic acid. Some examples of arylamino acids include ortho-, meta, and para-aminobenzoic acid. Someexamples of alkylaryl amino acids include ortho-, meta-, andpara-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid.

Non-naturally occurring amino acids also include derivatives ofnaturally occurring amino acids. The derivative of a naturally occurringamino acid may, for example, include the addition or one or morechemical groups to the naturally occurring amino acid.

For example, one or more chemical groups can be added to one or more ofthe 2′, 3′, 4′, 5′, or 6′ position of the aromatic ring of aphenylalanine or tyrosine residue, or the 4′, 5′, 6′, or 7′ position ofthe benzo ring of a tryptophan residue. The group can be any chemicalgroup that can be added to an aromatic ring. Some examples of suchgroups include hydroxyl, C₁-C₄ alkoxy, amino, methylamino,dimethylamino, nitro, halo (i.e., fluoro, chloro, bromo, or iodo), orbranched or unbranched C₁-C₄ alkyl, such as methyl, ethyl, n-propyl,isopropyl, butyl, isobutyl, or t-butyl.

Other examples of non-naturally occurring amino acids which arederivatives of naturally occurring amino acids include norvaline (Nva),norleucine (Nle), and hydroxyproline (Hyp).

The amino acids can be identical or different from one another. Basesare attached to the amino acid unit by molecular linkages. Examples oflinkages are methylene carbonyl, ethylene carbonyl and ethyl linkages.(Nielsen et al., Peptide Nucleic Acids-Protocols and Applications,Horizon Scientific Press, pages 1-19; Nielsen et al., Science 254:1497-1500.) One example of an amino acid residue of a PNA moiety isN-(2-aminoethyl)-glycine.

Further examples of PNA moieties include cyclohexyl PNA, retro-inversoPNA, phosphone PNA, propionyl PNA and aminoproline PNA. For adescription of these PNA moieties, see FIG. 5 of Nielsen et al., PeptideNucleic Acids-Protocols and Applications, Horizon Scientific Press,pages 1-19. FIG. 5 on page 7 of Nielsen et al. is hereby incorporated byreference.

PNA can be chemically synthesized by methods known in the art, e.g. bymodified Fmoc or tBoc peptide synthesis protocols. The PNA has manydesirable properties, including high melting temperatures (Tm), highbase-pairing specificity with nucleic acid and an uncharged molecularbackbone. Additionally, the PNA does not confer RNase H sensitivity onthe target RNA, and generally has good metabolic stability.

Peptide nucleic acids are also commercially available from AppliedBiosystems (Foster City, Calif., USA).

Additional nuclease resistant moieties are known in the art. Forexample, the DNA virus microRNA molecule comprises at least onemorpholino phosphoroamidate nucleotide moiety. Molecules comprisingmorpholino phosphoroamidate nucleotide moieties are generally referredto as morpholino (MF) nucleic acids. See structure 8 in FIG. 1. Heasman,Dev. Biol. 243, 209-214 (2002). Morpholino oligonucleotides arecommercially available from Gene Tools LLC (Corvallis, Oreg., USA).

In another example of a nuclease resistant moiety, the DNA virusmicroRNA molecule comprises at least one cyclohexene nucleotide moiety.Molecules comprising cyclohexene nucleotide moieties are generallyreferred to as cyclohexene nucleic acids (CeNA). See structure 10 inFIG. 1. Wang et al., J. Am. Chem. Soc. 122, 8595-8602 (2000), Verbeureet al., Nucleic Acids Res. 29, 4941-4947 (2001).

In a final example of a nuclease resistant moiety, the DNA virusmicroRNA molecule comprises at least one tricyclo nucleotide moiety.Molecules comprising tricyclo nucleotide moieties are generally referredto as tricyclo nucleic acids (tcDNA). See structure 9 in FIG. 1.Steffens et al., J. Am. Chem. Soc. 119, 11548-11549 (1997), Renneberg etal., J. Am. Chem. Soc. 124, 5993-6002 (2002).

In another embodiment, caps can be attached to one end, both ends,and/or between the ends of the molecule in order to increase nucleaseresistance of the DNA virus microRNA analogs or unmodified isolatednucleic acid microRNA molecules of the present invention described aboveto exonucleoses. Any cap known to those in the art for increasingnuclease resistance can be employed.

Examples of such caps include inverted nucleotide caps and chemicalcaps. Inverted nucleotide caps can be attached at the 5′ and/or 3′ end.Chemical caps can be attached to one end, both ends, and/or between theends of the molecule.

An inverted nucleotide cap refers to a 3′→5′ sequence of nucleic acidsattached to the DNA virus microRNA molecule or isolated nucleic acidmicroRNA molecules. There is no limit to the maximum number ofnucleotides in the inverted cap just as long as it does not interferewith binding of the molecule to its target mRNA. Any nucleotide can beused in the inverted nucleotide cap. Usually, the nucleotide cap is lessthan about forty nucleotides in length, preferably less than aboutthirty nucleotides in length, more preferably less than about twentynucleotides in length, and even more preferably less than about tennucleotides in length. Typically, the inverted nucleotide cap is onenucleotide in length. The nucleotide for the inverted cap is generallythymine, but can be any nucleotide such as adenine, guanine, uracil, orcytosine.

A chemical cap refers to any chemical group known to those in the artfor increasing nuclease resistance of nucleic acids. Example of suchchemical caps include hydroxyalkyl groups (alkyl hydroxides) oraminoalkyl groups (alkyl amines). Hydroxyalkyl groups are sometimesreferred to as alkyl glycoyl groups (e.g., ethylene glycol). Aminoalkylgroups are sometimes referred to as amino linkers.

The alkyl chain in the hydroxyalkyl group or aminoalkyl group can be astraight chain or branched chain. The minimum number of carbon atomspresent in the alkyl chain is one, preferably at least two, and morepreferably at least about three carbon atoms.

The maximum number of carbon atoms present in the alkyl chain is abouteighteen, preferably about sixteen, and more preferably about twelve.Typical alkyl groups include methyl, ethyl, and propyl. The alkyl groupscan be further substituted with one or more hydroxyl and/or aminogroups.

Some examples of amino linkers are shown in Table C. The amino linkerslisted in Table C lists are commercially available from TriLinkBiotechnologies, San Diego, Calif. TABLE C Amino Linkers from TriLinkBiotechnologies 2′-Deoxycytidine-5-C6 Amino Linker (3′ Terminus)2′-Deoxycytidine-5-C6 Amino Linker (5′ or Internal) 3′ C3 Amino Linker3′ C6 Amino Linker 3′ C7 Amino Linker 5′ C12 Amino Linker 5′ C3 AminoLinker 5′ C6 Amino Linker C7 Internal Amino Linker Thymidine-5-C2 AminoLinker (5′ or Internal) Thymidine-5-C6 Amino Linker (3′ Terminus)Thymidine-5-C6 Amino Linker (Internal)

Chimeric DNA virus microRNA molecules containing a mixture of any of themoieties mentioned above are also known, and may be made by methodsknown, in the art. See, for example, references cited above, and Wang etal, Proc. Natl. Acad. Sci. USA 96, 13989-13994 (1999), Liang et al.,Eur. J. Biochem. 269, 5753-5758 (2002), Lok et al., Biochemistry 41,3457-3467 (2002), and Damha et al., J. Am. Chem. Soc. 120, 12976-12977(2002).

The DNA virus microRNA molecules of the invention comprise at least ten,preferably at least thirteen, more preferably at least fifteen, and evenmore preferably at least twenty contiguous bases having the sequence ofa naturally occurring DNA virus microRNA molecule. In a preferredembodiment, the DNA virus microRNA molecules comprise the entiresequence of a DNA virus microRNA molecule, such as any one of the DNAvirus microRNA molecule sequences shown in Table A, Table A1 or TableA2.

The remaining bases in the molecule, if any, can be any modified orunmodified moiety described above. In one embodiment, the DNA virusmicroRNA molecule comprises at least one moiety which is aribonucleotide moiety or a deoxyribonucleotide moiety.

Any number of additional moieties, up to a maximum of forty moieties,having any base sequence can be added to the moieties comprising thecontiguous base sequence, as long as the total number of moieties in themolecule does not exceed fifty. The additional moieties can be added tothe 5′ end, the 3′ end, or to both ends of the contiguous sequence. Theadditional bases can include a sequence of bases at the 5′ end and/or asequence of bases at the 3′ end present in the hairpin precursor fromwhich the DNA virus microRNA is derived. In one embodiment, the hairpinprecursor sequence is any one of the hairpin precursor sequences shownin Table A, Table A1 or Table A2 or any fragment thereof.

For the contiguous bases mentioned above, up to thirty percent of thebase pairs may be substituted by wobble base pairs. As used herein,wobble base pairs refer to either: i) substitution of a cytosine with auracil, or 2) the substitution of an adenine with a guanine, in thesequence of the DNA virus microRNA molecule. These wobble base pairs aregenerally referred to as UG or GU wobbles. Table D shows the number ofcontiguous bases and the maximum number of wobble base pairs in the DNAvirus microRNA molecule. TABLE D Number of contiguous Bases and MaximumNumber of Wobble Bases No. of Contiguous Bases 10 11 12 13 14 15 16 1718 19 20 21 22 23 Max. 3 3 3 3 4 4 4 5 5 5 6 6 6 6 No. of Wob- ble BasePairs

Further, in addition to the wobble base pairs, up to ten percent, andpreferably up to five percent of the contiguous bases can be additions,deletions, mismatches or combinations thereof. Additions refer to theinsertion in the contiguous sequence of any moiety described abovecomprising any one of the bases described above. Deletions refer to theremoval of any moiety present in the contiguous sequence. Mismatchesrefer to the substitution of one of the moieties comprising a base inthe contiguous sequence with any of the above described moietiescomprising a different base.

The additions, deletions or mismatches can occur anywhere in thecontiguous sequence, for example, at either end of the contiguoussequence or within the contiguous sequence of the DNA virus microRNAmolecule. Typically, the additions, deletions or mismatches occur at theend of the contiguous sequence if the contiguous sequence is relativelyshort, such as, for example, from about ten to about fifteen moieties inlength. If the contiguous sequence is relatively long, such as, forexample, a minimum of sixteen contiguous sequences, the additions,deletions, or mismatches typically occur anywhere in the contiguoussequence.

For example, none or one of the contiguous bases may be additions,deletions, or mismatches when the number of contiguous bases is ten tonineteen; and a maximum of one or two additions, deletions, ormismatches are permissible when the number of contiguous bases is twentyto twenty-three.

Furthermore, no more than fifty percent, and preferably no more thanthirty percent, of the contiguous moieties contain deoxyribonucleotidebackbone units. Table E and F show the number of contiguous bases andthe maximum number of deoxyribonucleotide backbone units. TABLE E FiftyPercent of the Contiguous Moieties containing DeoxyribonucleotideBackbone Units No. of Contiguous Bases 10 11 12 13 14 15 16 17 18 19 2021 22 23 Max. No. of 5 5 6 6 7 7 8 8 9 9 10 10 11 11 DeoxyribonucleotideBackbone Units

TABLE F Thirty Percent of the Contiguous Moieties ContainingDeoxyribonucleotide Backbone Units No. of Contiguous Bases 10 11 12 1314 15 16 17 18 19 20 21 22 23 Max. No. of 3 3 3 3 4 4 4 5 5 5 6 6 6 6Deoxyribonucleotide Backbone Units

In another embodiment, in addition to the wobble base pairs and thefurther additions, deletions, and mismatches, described above, themoiety corresponding to position 11 in a naturally occurring DNA virusmicroRNA sequence can be an addition, deletion or mismatch.

Isolated MicroRNP

In another aspect, the invention provides an isolated microRNPcomprising any of the isolated nucleic acid sequences described above oranalogs of the DNA virus microRNAs described above.

Anti-DNA Virus MicroRNA Molecules

In another aspect, the invention provides an anti-DNA virus microRNAmolecule. The anti-DNA virus microRNA molecule may be any of theisolated nucleic acid sequences described above or analogs of the DNAvirus microRNAs described above, except that the sequence of bases ofthe anti-DNA virus microRNA molecule is complementary to the sequence ofbases in an isolated nucleic acid DNA microRNA sequence or analogs ofDNA virus microRNA molecules.

Examples of sequences of anti-DNA virus microRNA molecules is shown inTables G, G1 and G2. TABLE G EBV anti-microRNA Sequences Anti-microRNASequence Virus 5′ → 3′ EBV AACUCCGGGGCUGAUCAGGUUAUUCAAUUUCUGCCGCAAAAGAUA GUGUGCUUACACACUUCCCGUUA AGCACGUCACUUCCACUAAGAGCAAGGGCGAAUGCAGAAAAUA

TABLE G1 KSHV 8 anti-microRNA Sequences Anti-microRNA Sequence Virus 5′→3′ KSHV GCCACUCGGGGGGACAACACUA GCCACUCGGGGGGACAACACCAAGCGGGGUUUACGCAGCUGGGU UUACGCAGCUGCGUAUACCCAG CCGAUGGAUUAGGUGCUGCUGGCUCAACAGCCCGAAAACCAUCA CCGGCAAGUUCCAGGCAUCCUA CCUAGAGUACUGCGGUUUAGCUUCAGCUAGGCCUCAGUAUUCUA CGCUGCCGUCCUCAGAAUGUGA GUGUCACAUUCUGUGACCGCGACUUACACCCAGUUUCCUGUAAU GAGCGCCAGCAACAUGGGAUCA UCGGACACAGGCUAAGCAUUAACGUGCUCUCUCAGUCGCGCCUA

TABLE G2 HCMV anti-microRNA Sequences Anti-microRNA Sequence Virus 5′→3′ HCMV ACUCUCACGGGAAGGCUAGUUA CUACAAACUAGCAUUGUGGUGAUCUUUCCAGGUGUCUUCAACGA AGCCUGGAUCUCACCGUCACUU GCGGUGAAGAAGGGGAGGACGAAACGCUCUCGUCAGGCUUGUCA GACAUCGUCACACCUAUCAUAA CGGUCCGAGCCACUGAGCGGUUUCAUCCACCUGAACAGACCGCU CGCGGGAGCUGUCCAAGUGGAU CGCCCACGGUGCGGGCAGAAUC

The anti-DNA virus microRNA molecule can be modified as described abovefor DNA virus microRNA molecules. In one embodiment, the contiguousmoieties in the anti-DNA virus microRNA molecule are complementary tothe corresponding DNA virus microRNA molecule. The degree ofcomplementarity of the anti-DNA virus microRNA molecules are subject tothe restrictions described above for analogs of DNA virus microRNAmolecules, including the restriction relating to wobble base pairs, aswell as those relating to additions, deletions and mismatches.

In a preferable embodiment, if the anti-DNA virus microRNA moleculecomprises only unmodified moieties, then the anti-DNA virus microRNAmolecule comprises at least one base, in the at least ten contiguousbases, which is non-complementary to the DNA virus microRNA and/orcomprise a chemical cap.

In another preferable embodiment, if the at least ten contiguous basesin an anti-DNA virus microRNA molecule is perfectly complementary (i.e.,100%) to a DNA virus microRNA molecule, then the anti-DNA virus microRNAmolecule contains at least one modified moiety in the at least tencontiguous bases and/or comprises a chemical cap.

In yet another embodiment, the moiety in the anti-DNA virus microRNAmolecule at the position corresponding to position 11 of a naturallyoccurring DNA virus microRNA is non-complementary. The moiety in theanti-DNA virus microRNA molecule corresponding to position 11 of anaturally occurring DNA virus microRNA can be rendered non-complementaryby any means described above, including by the introduction of anaddition, deletion or mismatch, as described above.

Isolated

The nucleic acid molecule, DNA virus microRNA molecule or anti-DNA virusmicroRNA molecule is preferably isolated, which means that it isessentially free of other nucleic acids. Essentially free from othernucleic acids means that the nucleic acid molecule, DNA virus microRNAmolecule or anti-DNA virus microRNA molecule is at least about 90%,preferably at least about 95% and, more preferably at least about 98%free of other nucleic acids.

Preferably, the molecule is essentially pure, which means that themolecule is free not only of other nucleic acids, but also of othermaterials used in the synthesis and isolation of the molecule. Materialsused in synthesis include, for example, enzymes. Materials used inisolation include, for example, gels, such as SDS-PAGE. The molecule isat least about 90% free, preferably at least about 95% free and, morepreferably at least about 98% free of other nucleic acids and such othermaterials.

Utility

The DNA virus microRNA molecules and anti-DNA virus microRNA moleculesof the present invention have numerous in vitro, ex vivo, and in vivoapplications.

For example, the microRNA molecules and/or anti-microRNA molecules ofthe present invention can be introduced into a cell to study thefunction of the microRNA. Any DNA viral microRNA molecule and/oranti-DNA viral microRNA molecule mentioned above can be introduced intoa cell for studying their function.

In one embodiment, a microRNA in a cell is inhibited with a suitableanti-microRNA molecule. Alternatively, the activity of a microRNAmolecule in a cell can be enhanced by introducing into the cell anadditional microRNA molecule. The function of the microRNA can beinferred by observing changes associated with inhibition and/or enhancedactivity of the microRNA in the cell.

Thus, in one aspect of the invention, the invention relates to a methodfor inhibiting microRNP activity in a cell. The microRNP comprises a DNAvirus microRNA molecule. The microRNA molecule comprises a sequence ofbases complementary to the sequence of bases in a single strandedanti-DNA virus microRNA molecule. Any anti-DNA virus microRNA moleculecan be used in the method for inhibiting microRNP activity in a cell, aslong as the anti-DNA virus microRNA is complementary, subject to therestrictions described above, to the DNA virus microRNA present in themicroRNP.

The anti-DNA virus microRNA molecules of the present invention arecapable of inhibiting microRNP activity by binding to the DNA virusmicroRNA in the microRNP in a host cell. MicroRNP activity refers to thecleavage or the repression of translation of the target sequence. Thetarget sequence may be any sequence which is partially or perfectlycomplementary to the sequence of bases in a DNA virus microRNA. Thetarget sequence can be, for example, a viral or host messenger RNA.

For example, a DNA virus can produce a microRNA which is complementaryto a host derived target sequence that is beneficial to the host cellfor defending against the viral infection. The DNA virus microRNA, whichis packaged in a microRNP, will inhibit the beneficial effect of thetarget sequence. Accordingly, the introduction of the anti-DNA virusmicroRNA molecule inhibits the RNP activity, and thereby reduces harmfrom the virus.

Alternatively, a host cell can defend against a viral infection bytranscribing a gene which is harmful to the virus. For instance, thegene may induce the cell to undergo apoptosis, and therefore the gene isharmful to the virus. A DNA virus microRNA complementary to the targetsequence transcribed by the host cell is beneficial to the virus,because the DNA virus micro RNA (in a microRNP) will inhibit the abilityof the host cell to undergo apoptosis. Accordingly, the introduction ofDNA virus microRNA molecules promotes survival of the cell, therebyenhancing the infection.

The method for inhibiting microRNP activity in a cell comprisesintroducing into the cell a single-stranded anti-DNA virus microRNAmolecule. The anti-DNA virus microRNA molecule can be introduced into acell by any method described in the art. Some examples are describedbelow.

The cell can be any cell capable of being infected with a particular DNAvirus. Particular cells infected by a particular DNA virus are wellknown to those skilled in the art. For example, it is well known tothose in the art that EBV preferentially infects B lymphocytes.

The microRNA molecules or anti-microRNA molecules can be introduced intoa cell by any method known to those skilled in the art. For example, themolecules can be injected directly into a cell, such as bymicroinjection. Alternatively, the molecules can be contacted with acell, preferably aided by a delivery system.

Useful delivery systems include, for example, liposomes and chargedlipids. Liposomes typically encapsulate oligonucleotide molecules withintheir aqueous center. Charged lipids generally formlipid-oligonucleotide molecule complexes as a result of opposingcharges.

These liposomes-oligonucleotide molecule complexes orlipid-oligonucleotide molecule complexes are usually internalized incells by endocytosis. The liposomes or charged lipids generally comprisehelper lipids which disrupt the endosomal membrane and release theoligonucleotide molecules.

Other methods for introducing a microRNA molecule or an anti-microRNAinto a cell include use of delivery vehicles, such as dendrimers,biodegradable polymers, polymers of amino acids, polymers of sugars, andoligonucleotide-binding nanoparticles. In addition, pluoronic gel as adepot reservoir can be used to deliver the anti-microRNA oligonucleotidemolecules over a prolonged period. The above methods are described in,for example, Hughes et al., Drug Discovery Today 6, 303-315 (2001);Liang et al. Eur. J. Biochem. 269 5753-5758 (2002); and Becker et al.,In Antisense Technology in the Central Nervous System (Leslie, R. A.,Hunter, A. J. & Robertson, H. A., eds), pp. 147-157, Oxford UniversityPress.

Targeting of a microRNA molecule or an anti-microRNA molecule to aparticular cell can be performed by any method known to those skilled inthe art. For example, the microRNA molecule or anti-microRNA moleculecan be conjugated to an antibody or ligand specifically recognized byreceptors on the cell. For example, if the cell is a B lymphocyte, theantibody can be against the cell receptor CD19, CD20, CD21, CD23 or aligand to these receptors.

In another embodiment, the invention provides a method for treating aDNA virus infection is a mammal in need thereof. The method comprisesintroducing into the mammal an anti-DNA virus microRNA molecule. Theanti-DNA virus microRNA molecules can be introduced into the mammal byany method known to those in the art. For example, the above describedmethods for introducing the anti-DNA molecules into a cell can also beused for introducing the molecules into a mammal.

EXAMPLES Example 1 Materials and Methods

Cell lines and viruses. The EBV negative BL-41 and EBV positive BL41/95cells were described previously (Torsteinsdottir et al., Int. J. Cancer1989, 43:273) and were maintained in RPMI 1640 (Gibco) supplemented with10% FBS. BL41/95 but not BL-41 contained EBV, as confirmed by Westernblot analysis using antibodies against EBNA-1. For analysis of EBV miRNAexpression, we also cultured Hodgkin's lymphoma (HD) cells L540 andHD-MY-Z (EBV negative) and RPMI 6666 (EBV positive) and the Burkitt'slymphoma (BL) cells Ramos (EBV negative), Ous and Mutu (EBV positive),and EBV positive Marmoset B95-8 cells that produce infectious B95-8viral particles. These cell lines were also maintained in RPMI 1640(Gibco) supplemented with 10% FBS. The KSHV positive BCBL1 cell line wasdescribed previously (Renne et al. Nat. Med. 1996, 2:342-346) and wasmaintained in RPMI 1640 (Gibco) supplemented with 10% FBS. For the KSHVstudies, to induce viral replication, a total of 5×10⁶ BCBL1 cells wereinduced with 20 ng of phorbol-12-tetradecanoate-13-acetate (TPA)/ml andRNA was isolated 24, 48 and 72 h after TPA treatment. Primary humanforeskin fibroblasts were cultured in MEM (GIBCO) supplemented with 10%FCS, 10 U/ml moronal, and 10 μg/ml neomycin sulphate. Cells at 90%confluency were infected with HCMV strain VR1814 at 5 PFU/cell andharvested when a strong cytopathic effect was visible, usually at about4-5 days post-infection.

RNA preparation, cloning procedure and Northern blot analysis. Total RNAextraction was performed as described previously (Lagos-Quintana et al.,Curr. Biol. 2002, 12:735). RNA size fractionation and cloning procedurehave also been described. Northern blot analysis was performed asdescribed (Lagos-Quintana et al., Curr. Biol. 2002, 12:735) loading 30μg or 15 μg of total RNA per lane and using 5′ ³²P-radiolableledoligodeoxynucleotides complementary to the mRNA sequence. For the EBVstudies, equal loading of the gels was confirmed by ethidium bromidestaining of the tRNA band or by reprobing the blot for U6 snRNA using³²P-labeled 5′GCAGGGGCCATGCTAATCTTCTCTGTATCG oligodeoxynucleotide. Blotswere stripped and reprobed several times. Complete stripping of the blotwas confirmed by phosphorimaging of the membrane before reprobing.

DNA Sequencing of small RNA cDNA libraries. Bacterial colonies werepicked into 96 well plates filled with 20 μl sterile water per well,then diluted 1:1 into a second 96 well plate containing 10 μl PCRcocktail (2 μl 10× Sigma JumpStart PCR buffer, 2 μl 2 mM deoxynucleosidetriphosphate mixture, 0.4 μl each 10 μM M13 universal and reverseprimers, 0.35 μl 1 U/μl JumpStart REDAccuTaq DNA polymerase (Sigma), and4.85 μl water. The PCR cycling program consisted of 1′30″ at 94° C.,followed by 30 cycles of 94° C., 30″; 57° C., 30″; 72° C., 3′30″,conditions which largely deplete the primers and deoxynucleotides,obviating the requirement for reaction cleanup prior to sequencing.After diluting the PCR products with 30 μl water, 3 μl was added towells of a 96 well plate containing 17 μl sequencing cocktail consistingof 1 μl 2.5× BigDye Terminator v3.1 Cycle Sequencing Kit premix, 1.75 μl5× buffer and 14.25 μl water, and sequencing reactions were carried outfor 25 cycles (96° C., 10″; 50° C., 5″; 60° C., 4′). The reactionproducts were precipitated with 50 μl 100% ethanol/2 μl 3M NaOAc (pH4.8), pellets were rinsed with 70% ethanol, and after the addition of 10μL1 Hi-Di Formamide (Applied Biosystems) and denaturing at 94° C. for 10min, samples were loaded onto an ABI 3730×l sequencer.

miRNA target prediction. We first obtained the 3′ UTR sequences for20,153 transcripts in the human genome using Ensmart (Kasprzyk et al.,Genome Res. 2004, 14:160) as well as the sequences of 175 mature humanmRNAs from the RFAM mRNA registry (Griffiths-Jones, Nucleic Acids Res.,2004, 32:D109). miRanda (Enright et al., Genome Biol., 2003, 5:RI, 1)was used to identify mRNA binding sequences in the 3′ UTR sequences. Thethresholds used for this scan were S:90 and .G: −17 kcal/mol. Targetsthat were in the 90th percentile of the raw alignment scores wereselected as candidate mRNA targets.

Example 2 Identification of EBV Encoded microRNAs

We examined a large DNA virus of the Herpes family, Epstein barr virus(EBV) which preferentially infects human B cells. We cloned the smallRNAs from a Burkitt's lymphoma cell line latently infected with EBV.Surprisingly, we found 4% of the cloned small RNAs originated from EBV(Tables 1 and 2). Table 1. Composition of small RNA cDNA librariesprepared from non-infected (−) and DNA virus-infected human cell linesaccording to sequence annotation. The annotation was based oninformation from GenBank (http://www.ncbi.nih.gov/Genbank/index.html), adataset of human tRNA sequences(http://rna.wustl.edu/GtRDB/Hs/Hs-seqs.html), a dataset of human andmouse sn/snoRNA sequences (http://mbcr.bcm.tmc.edu/smallRNA/Database), adatabase of microRNAs (http://www.sanger.ac.uk/Software/Rfam/microRNA/),predictions of microRNAs (35), and the repeat element annotation of theHG16 human genome assembly from UCSC (http://genome.cse.ucsc.edu). Thetotal number of cloned sequences is indicated in parentheses at thebottom line of the table. Sequences that mapped to the human genomeallowing up to two mismatches but could not be assigned a specific typewere classified as Not annotated; those that did not match to the genomewith more than 3 mismatches were classified as Not matched. BL-41 HumanCell Line Type — EBV rRNA   37.00   41.92 tRNA    5.32    4.72 microRNA  44.36   33.94 Repeat    1.62    0.98 Other ncRNA^(a)    4.33    5.80mRNA    4.11    5.39 Viral^(b)    0    4.15 Not annotated    2.26   2.23 Not matched.    0.99    0.88 (No. seq.) (2216) (1930)^(a)This includes snRNAs and snoRNAs and other known small cytoplasmicnon-coding RNAs.^(b)The annotation for viral sequences is based on EBV B95-8 (GenBankV01555).

TABLE 2 Small RNA sequences derived from viral sequence. The position ofthe small RNA sequence is given relative to the viral genome sequencesspecified in Table 1 above. Small RNA Sequence Size range Virus 5′→ 3′Clones (nt) Position, Orientation EBV UAACCUGAUCAGCCCCGGAGUU 2 21-2253762-53783, + AAAUUCUGUUGCAGCAGAUAGC 3 22 55141-55162, +UAUCUUUUGCGGCAGAAAUUGAA 50 20-23 55176-55198, + UAACGGGAAGUGUGUAAGCACAC23 19-23 55256-55278, + UCUUAGUGGAAGUGACGUGGU 1 21 151640-151660, +UAUUUUCUGCAUUCGCCCUUGC 2 22 153205-153226, +

Most of the EBV sequences were cloned more than once and the analysis ofthe genomic sequence flanking the cloned RNAs suggested fold-backstructures characteristic of microRNAs genes. The EBV microRNAsoriginated from 5 different dsRNA precursors that are clustered in tworegions of the EBV genome (FIGS. 2A and B).

The EBV microRNAs were all readily detectable by Northern blotting,including the approximately 60-nt fold-back precursor for 3 of the 5microRNAs (FIG. 2C). The first microRNA cluster is located within themRNA of the BHFR1 gene encoding a distant Bcl-2 homolog, and we refer tothese three microRNAs as miR-BHRF1-1 to miR-BHRF1-3.

miRBHFR1-1 is located in the 5′ UTR and miR-BHFR1-2 and -3 arepositioned in the 3′ UTR of the BHRF1 μmRNA. Structurally similarmicroRNA gene organization has been observed for some mammalianmicroRNAs that flank open reading frames in expressed sequence tags. Theother EBV microRNAs cluster in intronic regions of the BART gene, and werefer to them as miR-BART1 and miR-BART2. Since microRNAs function inRNA silencing pathways either by targeting mRNAs for degradation or byrepressing translation, we identified new viral regulators of hostand/or viral gene expression.

Example 3 Predicated Target for Epstein Barr Virus Encoded microRNA

EBV latently infected cells can be found in three different latentstages (I to III, FIG. 2A) that are characterized by the expression ofvarious subsets of the latent genes: six nuclear antigens (EBNAs 1, 2,3A, B, C, and EBNA-LP), three latent membrane proteins (LMPs 1, 2A and2B), two non-coding RNAs (EBERs 1 and 2) and transcripts from the BamHIA region (BARTs/CSTs) whose coding capacity is still controversial.

We isolated our small RNAs from a latent-stage-Ill EBV cell line thatexpresses all latent genes. In order to address if the expression of theEBV microRNAs is coupled with a specific latent stage, we probed for EBVmicroRNA expression in immortalized cell lines which are in differentstages of latency, including Hodgkin's lymphoma (HD, latency II),Burkitt's lymphoma (BL) latency stage I cells, and virus-producingmarmoset monkey lymphocytes B95-8 (latency III, with a fraction of 3 to10% of cells expressing lytic stage antigens) (FIG. 2D).

BART microRNAs were detected in all latent stages consistent with thereported expression of BART during every stage of EBV infection.However, BART microRNA expression was elevated by about 10-fold in thevirus producing marmoset cell line (FIG. 2D, lane 9, rows 5 and 6).Although several studies have attempted to identify proteins encodedfrom the different spliced transcripts of BART, the function of thisregion remains unknown. Our findings will help to assign a function tothe BART region.

The expression pattern of BHRF1 microRNAs is dependent on the EBVlatency stage. While cell lines in stage II and III expressed BHRF1microRNAs (FIG. 2D, lanes 5-6), only one of the two stage I cell linesexpressed BHRF1 microRNAs (FIG. 2D, lanes 7, 8). Latency I cell linesare thought to express only EBNA 1, the EBERs and the BARTs.

The expression of a transcript deriving from the BHRF1 region in one ofthe latency stage I cell lines as well as its expression in stage IIcell lines, suggests a new latency stage I/II promoter upstream of theknown latency stage I/II Qp promoter (FIG. 2A). A new subdivision oflatency I stages may have to be introduced to distinguish between BHRF1microRNA expressing cell lines in latency I.

Although BHRF1 protein is only detected in lytic stage, latent stage EBVtranscripts encompassing the BHRF1 region were observed previously. Itis likely that the microRNAs BHRF1-1 to 3 are also expressed duringlytic stage along with the BHRF1 protein. The high-level transcriptionof BHRF1 during the lytic cycle may exceed the cellular microRNAprocessing capacity and unprocessed transcripts could then betranslated.

To identify targets for EBV microRNAs, we used a computational methodrecently developed for prediction of Drosophila microRNAs targets(Enright et al., Genome Biol., 2003, 5:RI, 1). A set of approximately20,000 non-redundant human 3′ UTRs and the genome sequence of EBV weresearched for potential microRNA binding sites. The top scoring hits forwhich a gene function annotation was available, are listed in Table 3.The majority of predicted host cell targets have more than one bindingsite for the viral microRNA, and approximately 50% of these areadditionally targeted by one or several host cell microRNAs. MultiplemicroRNA binding sites are believed to act synergistically and increasetargeting efficiency in a cooperative non-linear fashion. TABLE 3Predicted host cell target mRNAs of EBV microRNA. The gene name isindicated as recommended by HUGO, and the gene function annotation wasextracted from Ensemble. The number of predicted microRNA binding sitesin the 3′ UTR of the target gene (NS) and a percentile score ranking thetarget site predictions (%-ile) are indicated. If human microRNAs arealso predicted to bind to a putative EBV microRNA regulated target, itis indicated in the last column. The predicated human microRNA bindingsites are also conserved in the orthologous mRNAs in mouse. EBV microRNAGene ID Proposed function NS %-ile Human miRNA Apoptosis, cellproliferation BART1, BCL2 Apoptosis regulator Bcl-2 3, 1 100, miR-217,miR-140 BHRF1-2 98 BHRF1-1 P53 Tumor suppressor P53 2 98 BHRF1-1 E2F1Retinoblastoma Binding protein 3, Transcription factor E2F-1 2 98miR-20, miR-106 Transcription regulation BART1 HIC2 Hypermethylated inCancer 2 Protein 2 99 BART1 ZNF177 Zinc Finger protein 177 4 100 BART2UBN1 Ubinuclein 1 3 100 BHRF1-1 CBFA2T2 Myeloid Translocationgene-related protein 1 3 100 miR-301 BHRF1-3 NSEP1 Y Box Binding protein1 94 miR-95, miR-216, miR-136 BHRF1-3, TGIF 5′-TG-3′ Interacting factor,Homeobox protein TGIF 1, 1 97 miR-194 BART2 97 Immune response BART2LRBA Lipopolysaccharide-responsive and beige like protein, BCL8 4 99miR-15a, miR-146 Homolog miR-29a BHRF1-1 LILRB5 Leukocyte immunoglobulinreceptor, subfamily B, member 5 2 100 BHRF1-3 PRF1 Perforin 1 precursor1 99 Signal transduction BART1 CXCL12 Stromal cell derived factor 1precursor, Pre-B growth 3 100 miR-106, miR-135 Stimulating factormiR-197 BART2 GAB2 GRB2-Associated Binding Protein 2 4 100 miR-155 BART2TNFRSF1A Tumor Necrosis Factor Receptor Superfamily member 1A 2 99BHRF1-2 PIK3R1 Phosphatidylinositol 3-kinase regulatory Alpha Subunit 192 let-7b BHRF1-2, B7RP-1 B7 homolog, ICOS ligand precursor 1, 3 97,miR-155 BART2 99 BHRF1-3 CXCL11 Small inducible cytokine B11 precursor,I-TAC 3 100 Chromosome organization BHRF1-2 CENPA Centromere Protein A 198 miR-16

Several of the predicted viral microRNA targets are prominent regulatorsof cell proliferation and apoptosis, which are presumably important forgrowth control of the infected cells. microRNA modulation of cellproliferation also provides new leads for studying the association ofEBV with several cancerous malignancies. Another important group of EBVmicroRNA targets are B-cell specific chemokines and cytokines, which areimportant for leukocyte activation and/or chemotaxis. Down-regulation ofthese genes presumably contributes to escape of EBV-infected B cellsfrom activated cytotoxic T cells. Additional targets includetranscriptional regulators and components of signal transductionpathways that are critical for maintaining or switching between EBVlytic and latent stages.

Example 4 EBV Encoded microRNA miR-BART2 Targets Virally Encoded DNAPolymerase BALF5

One of the EBV-encoded microRNAs, miR-BART2, is capable of targeting thevirally encoded DNA polymerase BALF5 for degradation (FIG. 3). miR-BART2is transcribed anti-sense to the BALF5 transcript and is thereforeperfectly complementary to the BALF5 3′ UTR and able to subject thismRNA for degradation. Similarly, the clustered miRBHRF1-2 and -3 arecomplementary to the transcript encoding the lytic gene BFLF2 (FIG. 2A),whose function is currently unknown. The down-regulation of lytic genesby viral microRNAs may contribute to establishment and maintenance oflatent infection.

Example 5 Identification of KSHV Encoded microRNAs

The role of Kaposi's sarcoma-associated herpesvirus (KSHV) in variouslymphomas is firmly established. To identify KSHV microRNAs, we clonedthe small RNAs from a body cavity based lymphoma (BCBL) cell line,latently infected with KSHV. We found that up to 21% of the total clonedsmall RNAs (34% of the cloned cellular mRNAs), originated from KSHV(Tables 4 and 5). Table 4. Composition in percentage of small RNA cDNAlibraries prepared from KSHV-infected human cell line according tosequence annotation. The annotation was based on information fromGenBank (http://www.ncbi.nih.gov/Genbank/index.html), a dataset of humantRNA sequences (http://rna.wustl.edu/GtRDB/Hs/Hs-seqs.html), a datasetof human and mouse sn/snoRNA sequences(http://mbcr.bcm.tmc.edu/smallRNA/Database), a database of mRNAs(http://www.sanger.ac.uk/Software/Rfam/mirna/), predictions of mRNAs,and the repeat element annotation of the HG16 human genome assembly fromUCSC (http://genome.cse.ucsc.edu). The total number of cloned sequencesis indicated in parentheses at the bottom line of the table. Sequencesthat mapped to the human genome allowing up to two mismatches but couldnot be assigned a specific type were classified as “Not annotated”. Theannotation for viral sequences is based on the published genomicsequence of KSHV BC-1 (GenBank U75698). Composition of small RNAs cDNAlibrary BCBL1 Type (%) rRNA    3.22 tRNA    4.78 sn/sno-RNA    0.29miscRNA    2.14 Repeat    2.24 mRNA    1.75 miRNA   61.60 Viral   20.96Not annotated    3.02 (No. seq.) (1026)

TABLE 5 Small RNA sequences derived from KSHV. The position of the smallRNA sequence is given relative to the viral genome sequence specified inTable 4. No. Small RNA sequence (5′ to 3′) Seq Position, orientationUAGUGUUGUCCCCCCGAGUGGC 36 117971-117991, − UGGUGUUGUCCCCCCGAGUGGC 39117971-117991, − ACCCAGCUGCGUAAACCCCGCU 2 119338-119359, −CUGGGUAUACGCAGCUGCGUAA 20 119304-119325, − CCAGCAGCACCUAAUCCAUCGG 14120796-120817, − UGAUGGUUUUCGGGCUGUUGAG 9 120765-120786, −UAGGAUGCCUGGAACUUGCCGGU 5 121266-121287, − UAGAAUACUGAGGCCUAGCUGA 12121417-121438, − AGCUAAACCGCAGUACUCUAGG 34 121455-121476, −UCGCGGUCACAGAAUGUGACA 12 121546-121566, − UCACAUUCUGAGGACGGCAGCGA 2121586-121608, − AUUACAGGAAACUGGGUGUAAGC 12 121889-121910, −UGAUCCCAUGUUGCUGGCGCU 13 120359-120380, − UUAAUGCUUAGCCUGUGUCCGA 4120580-120601, − UAGGCGCGACUGAGAGAGCACG 1 119945-119966, −

Most of the KSHV sequences were cloned more than once and the analysisof the genomic sequence flanking the cloned RNAs suggested fold-backstructures characteristic of microRNA genes. The KSHV microRNAsoriginated from 10 different dsRNA precursors that are all clustered inthe same region of the KSHV genome (FIGS. 4A and 4B).

The KSHV microRNAs were designated miR-K1 to miR-K10. The cluster islocated within the mRNA of the K12 gene encoding a protein namedKaposin, which possesses some oncogenic properties. Interestingly,miR-K1 is located within the coding sequence of K12. Previous reportssuggest that the K12 coding sequence region is complex and encodesseveral proteins named Kaposin A, B, and C (see FIG. 4A).

We also identified two isoforms of miR-K1, i.e. miR-K1a and miR-K1b,which differ by one nucleotide at position 2 (see Table 6). MiR-K1acorresponds to the sequenced genome present in BCBL1 cells. MiR-K1bappears to be derived from a sequence isolated from a primary effusionlymphoma (PEL) tumor. Thus, two difference viral genomes orquasi-species may be present in the BCBL1 cell line. MiR-K2 to miR-K10are located in the intronic region of a longer transcript encoding K12whose promoter is located upstream of the ORF 72 (see FIG. 4A).

We next investigated whether KSHV mRNAs are differentially regulatedupon induction of the lytic cycle. BCBL1 cells harbor replicationcompetent KSHV. Upon treatment with TPA, these cells undergo thecomplete program of KSHV gene expression, resulting ultimately in viralreplication and the release of mature virions.

We isolated total RNA after various times of TPA treatment and probedfor KSHV mRNAs expression by Northern blot. Only miR-K1a expression wasinduced upon treatment, whereas mRNAs in the intronic regions, such asmiR-K6 and miR-K7, where not affected (FIG. 5). This indicates thatmiR-K1a and miR-K2 to K10 may originate from different primarytranscripts (FIG. 4A).

The identification of mRNAs in the genome of KSHV will provide newinsights in the understanding of the oncogenic properties of the virus.TABLE 6 KSHV miRNAs mature and precursor sequences. In bold the matureform, underlined the non-functional star sequence that was cloned formiR-K2 and miR-K6. KSHV miRNA microRNA sequence (5′ to 3′) Hairpinprecursor sequence (5′ to 3′) miR-K1a UAGUGUUGUCCCCCCGAGUGGCCUGGAGGCUUGGGGCGAUACCACCACU CGUUUGUCUGUUGGCGAUUAGUGUUG UCCCCCCGAGUGGCCAGmiR-K1b UGGUGUUGUCCCCCCGAGUGGC CUGGAGGCUUGGGGCGAUACCACCACUCGUUUGUGUGUUGGCGAUUGGUGUUG UCCCCCCGAGUGGCCAG miR-K2 *AGCCAGCUGCGUAAACCCCGCU GGGUCUACCCAGCUGCGUAAACCCCGCCUGGGUAUACGCAGCUGCGUAA UGCGUAAACACAGCUGGGUAUACGCA GCUGCGUAAACCC miR-K35p CCAGCAGCACCUAAUCCAUCGG CUUGUCCAGCAGCACCUAAUCCAUCG 3pUGAUGGUUUUCGGGCUGUUGAG GCGGUCGGGCUGAUGGUUUUCGGGCU GUUGAGCGAG miR-K4UAGGAUGCCUGGAACUUGGCGGU UGACCUAGGUAGUCCCUGGUGCCCUAAGGGUCUAGAUCAAGCAGUUAGGAUGC CUGGAACUUGCCGGUCA miR-K5 5pAGGUAAACCGCAGUACUCUAGG AUAACUAGCUAAACCGCAGUACUCUA 3pUAGAAUACUGAGGGCUAGCUGA GGGCAUUGAUUUGUUAGAUAGAAUAC UGAGGCCUAGCUGAUUAUmiR-K6 UCACAUUCUGAGGACGGCAGCGA GGCUAUCACAUUCUGAGGACGGCAGCGACGUGUGUCUAACGUCAACGUCGCG * UGGCGGUCACAGAAUGUGAGA GUCACAGAAUGUGACACCmiR-K7 AUUACAGGAAACUGGGUGUAAGC GGAUUACAGGAAACUGGGUGUAAGCUGUACAUAAUCGCCGGCAGGACCUGUU UCCUGCAAGCCUCGU miR-K8 UGAUCCCAUGUUGCUGGCGCUGCGUUGAGCGCCACCGGACGGGGAUU UAUGCUGUAUCUUACUACCAUGAUCCCAUGUUGCUGGCGCUCACGG miR-K9 UUAAUGCUUAGCCUGUGUCCGACGCUUUGGUCACAGCUUAAACAUUUC UAGGGCGGUGUUAUGAUCCUUAAUGCUUAGCCUGUGUCCGAUGCG MiR-K10 UAGGCGCGAGUGAGAGAGCACGCGCGCACUGGCUCACUAACGCCCCGCU UUUGUCUGUUGGAAGCAGCUAGGCGC GACUGAGAGAGCACGCG

Example 6 Identification of HCMV Encoded microRNAs

HCMV is a ubiquitous member of the β-herpesvirus family. Although HCMVinfection of healthy children and adults is normally asymptomatic, itremains a leading cause of birth defects and an important cause ofmortality in immunocompromised individuals.

Small RNAs were cloned from primary human foreskin fibroblasts lyticallyinfected with HCMV clinical strain VR1814. We cloned 424 small RNAsderiving from the virus genome in HCMV infected cells. Of these, 171sequences were cloned once, and were dispersed throughout the genome;the 253 remaining sequences were cloned multiple times and analysis ofthe genomic sequences flanking these suggested structures characteristicof mRNAs (Tables 7 and 8 and FIG. 6).

Four miRNAs were located in the UL region of the genome, and fivederived from the US region. Interestingly, five mRNAs, miR-UL3, miR-UL4,miR-US3, miR-US4 and miR-US5 are transcribed on the complementary strandto known open reading frames (ORFs) (FIG. 6A). These five mRNAs may beinvolved in the cleavage of the complementary transcripts, as previouslydescribed for EBV miR-BART2 and the DNA polymerase BALF5. UL114 is ahomolog of the mammalian uracyl-DNA glycosylase and has been shown to berequired for efficient viral DNA replication. UL150 is an ORF that ispresent in the clinical strains of HCMV, but not in the laboratorystrains.

The other four mRNAs are either located in intergenic regions (miR-UL1,miR-US1 and miR-US2) or in an intronic region (miR-UL2). It isinteresting to note that miR-UL2 is located in the intron of UL36, whichhas been described as an inhibitor of apoptosis that suppressescaspase-8 activation. Table 7. Composition in percentage of small RNAcDNA libraries prepared from HCMV-infected human cell line according tosequence annotation (see Table 4). The annotation for viral sequences isbased on the published genomic sequence of HCMV FIX-BAC isolate VR1814(Genbank AC146907). Fibroblasts HCMV Type (%) rRNA + tRNA   44.72 OtherncRNA    3.12 mRNA    4.51 Repeat    5.61 miRNA   20.41 Viral   17.88Not annotated    3.75 (No. seq.) (2371)

TABLE 8 HCMV miRNAs mature and precursor sequences In bold the matureform, underlined the non-functional star sequence. Position of the Seq.precursor, HCMV miRNA Mature sequence No. Hairpin precursor sequenceorientation

miR-UL1 UAACUAGCCUUCCCGUGAGA 101 CCUGUCUAACUAGCCUUCCCGUGAGAGUUUAUG27644-27711, + AACAUGUAUCUCACCAGAAUGCUAGUUUGUAGA GG miR-UL1*UGAGCAGAAUGCUAGUUUGUAG 11 miR-UL2 UCGUUGAAGACACCUGGAAAGA 9CCACGUCGUUGAAGACACCUGGAAAGAGGACGU 49495-49569, 31UCGCUCGGGCACGUUCUUUCCAGGUGUUUUCAAC GUGCGUGG miR-UL3AAGUGACGGUGAGAUGCAGGCU 22 GACAGCCUCGGGAUCACAUGGUUACUCAGCGUCU164118-164184, + GCCAGGGUAAGUGACGGUGAGAUCCAGGCUGUC miR-UL4UCGUCCUCCCCUUCUUCACCG 5 AGCAGGUGAGGUUGGGGCGGACAACGUGUUGCG N.C. †GAUUGUGGCGAGAACGUCGUCCUCCCCUUCUUC ACCGCC 196991-197056, + miR-US1UGACAAGCCUGACGAGAGCGU 4 UGAACGCUUUCGUCGUGUUUUUCAUGCAGCUUUUACAGACCAUGACAAGCCUGACGAGAGCGUUCA miR-US2 UUAUGAUAGGUGUGACGAUGUC 46GGAGGCUUUCGCCACACCUAUCCUGAAAGCGUUG 197120-197184, +CAUUCUUUAUGAUAGGUGUGACGAUGUCUUU miR-US3 AACCGCUCAGUGGCUCGGACC 37UGUGAACCGCUCAGUGGCUCGGACCGCCGGCUG 216177-216246, −UUUGUGCGCACCGGUCCGAACGCUAGGUCGGUUC UCA miR-US4-5p AGCGGUCUGUUCAGGUGGAUGA11 CGGUUAGCGGUCUGUUCAGGUGGAUGAGGGCU 216379-216468, −CUUCACGGUCGGGCACUCGGCUGUGCCUGUCAUC CACUUGGAGAGCUCCCGCGGUCCG miR-US4-3pAUCCACUUGGAGAGCUCCCGCGG 3 miR-US5 GAUUGUGCCCGGACCGUGGGCG 4CACGGUUGAUUGUGCCCGGACCGUGGGCGCGA 221403-221472, −CGAAACCCACCGUCACGGUCCGAGCACAUCCAAA CGUG

Positions are given relative to the laboratory strain AD169, which isfully annotated (Genbank NC_(—001347))†miR-UL4 is the only HCMV miRNA that is not conserved between the FIXstrain used here and the AD 169 strain. It is located opposite to thegene ULISO, and the precursor location relative to the published HCMVFIX-BAG sequence (Genbank AC 146907) is 34630-34701 (−)

1-68. (canceled)
 69. An isolated nucleic acid molecule comprising any one of the sequences of a DNA virus microRNA shown in Table A2.
 70. An isolated nucleic acid molecule according to claim 69, wherein the microRNA is incorporated into a vector.
 71. An isolated nucleic acid molecule according to claim 69, wherein the DNA virus microRNA is part of a hairpin precursor sequence or fragment thereof.
 72. An isolated nucleic acid molecule according to claim 71, wherein the hairpin precursor sequence is incorporated into a vector.
 73. An isolated nucleic acid molecule according to claim 69, wherein the nucleic acid molecule is a DNA molecule.
 74. An isolated nucleic acid molecule according to claim 69, wherein the nucleic acid molecule is a RNA molecule.
 75. An isolated nucleic acid molecule according to claim 69, wherein the nucleic acid molecule consists of the DNA virus microRNA.
 76. An isolated nucleic acid molecule according to claim 71, wherein the nucleic acid molecule consists essentially of the DNA virus hairpin precursor sequence.
 77. An isolated nucleic acid molecule according to claim 71, wherein the DNA virus hairpin precursor sequence is any one of the sequences shown in Table A2.
 78. An isolated nucleic acid molecule according to claim 69, further comprising a sequence of bases at the 5′ end and/or a sequence of bases at the 3′ end present in a hairpin precursor from which the DNA microRNA is derived.
 79. An isolated nucleic acid molecule according to claim 78, wherein the DNA virus hairpin precursor sequence is any one of the sequences shown in Table A2 or any fragment thereof.
 80. An isolated single stranded DNA virus microRNA molecule comprising a minimum of ten moieties and a maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit wherein: at least ten contiguous bases have the same sequence as any one of the sequence of bases in a DNA virus microRNA molecule shown in Table A2, except that up to thirty percent of the bases pairs may be wobble base pairs, and up to 10% of the contiguous bases are additions, deletions, mismatches, or combinations thereof; and no more than fifty percent of the contiguous moieties contain deoxyribonuleotide backbone units.
 81. A molecule according to claim 80, further comprising a sequence of bases at the 5′ end and/or a sequence of bases at the 3′ end present in a hairpin precursor from which the DNA microRNA is derived.
 82. A molecule according to claim 81, wherein the hairpin precursor is any one of the hairpin precursor sequences shown in Table A2 or any fragment thereof.
 83. A molecule according to claim 80, wherein the molecule is modified for increased nuclease resistance.
 84. An isolated single stranded anti-DNA virus microRNA molecule comprising a minimum of ten moieties and a maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, each base forming a Watson-Crick base pair with a complementary base wherein: at least ten contiguous bases have a sequence complementary to a contiguous sequence of bases in the sequence of bases in any one of the DNA virus microRNA molecule shown in Table A2, except that up to thirty percent of the base pairs may be wobble base pairs, and up to 10% of the contiguous bases are additions, deletions, mismatches, or combinations thereof; no more than fifty percent of the contiguous moieties contain deoxyribonuleotide backbone units; and the molecule is capable of inhibiting microRNP activity.
 85. A molecule according to claim 84, wherein the moiety in the molecule at the position corresponding to position 11 of the microRNA is non-complementary.
 86. A molecule according to claim 84, wherein up to 5% of the contiguous moieties are non-complementary to the contiguous sequence of bases in the DNA virus microRNA.
 87. A molecule according to claim 86, wherein non-complementary moieties are additions, deletions, mismatches, or combinations thereof.
 88. A molecule according to claim 84 having the anti-DNA virus microRNA sequence shown in Table G.
 89. A molecule according to claim 84, wherein at least one of the moieties is a deoxyribonucleotide.
 90. A molecule according to claim 89, wherein the deoxyribonucleotide is a modified deoxyribonucleotide moiety.
 91. A molecule according to claim 90, wherein the modified deoxyribonucleotide is a phosphorothioate deoxyribonucleotide moiety.
 92. A molecule according to claim 90, wherein the modified deoxyribonucleotide is N′3-N′5 phosphoroamidate deoxyribonucleotide moiety.
 93. A molecule according to claim 84, wherein at least one of the moieties is a ribonucleotide moiety.
 94. A molecule according to claim 93, wherein at least one of the moieties is a modified ribonucleotide moiety.
 95. A molecule according to claim 94, wherein the modified ribonucleotide is substituted at the 2′ position.
 96. A molecule according to claim 95, wherein the substituent at the 2′ position is a C₁ to C₄ alkyl group.
 97. A molecule according to claim 96, wherein the alkyl group is methyl.
 98. A molecule according to claim 96, wherein the alkyl group is allyl.
 99. A molecule according to claim 95, wherein the substituent at the 2′ position is a C₁ to C₄ alkoxy —C₁ to C₄ alkyl group.
 100. A molecule according to claim 99, wherein the C₁ to C₄ alkoxy —C₁ to C₄ alkyl group is methoxyethyl.
 101. A molecule according to claim 94, wherein the modified ribonucleotide has a methylene bridge between the 2′-oxygen atom and the 4′-carbon atom.
 102. A molecule according to claim 84, wherein at least one of the moieties is a peptide nucleic acid moiety.
 103. A molecule according to claim 84, wherein at least one of the moieties is a 2′-fluororibonucleotide moiety.
 104. A molecule according to claim 84, wherein at least one of the moieties is a morpholino phosphoroamidate nucleotide moiety.
 105. A molecule according to claim 84, wherein at least one of the moieties is a tricyclo nucleotide moiety.
 106. A molecule according to claim 84, wherein at least one of the moieties is a cyclohexene nucleotide moiety.
 107. A molecule according to claim 84, wherein the molecule comprises at least one modified moiety for increased nuclease resistance.
 108. A molecule according to claim 107, wherein the nuclease is an exonuclease.
 109. A molecule according to claim 108, wherein the molecule comprises at least one modified moiety at the 5′ end.
 110. A molecule according to claim 108, wherein the molecule comprises at least two modified moieties at the 5′ end.
 111. A molecule according to claim 108, wherein the molecule comprises at least one modified moiety at the 3′ end.
 112. A molecule according to claim 108, wherein the molecule comprises at least two modified moieties at the 3′ end.
 113. A molecule according to claim 108, wherein the molecule comprises at least one modified moiety at the 5′ end and at least one modified moiety at the 3′end.
 114. A molecule according to claim 108, wherein the molecule comprises at least two modified moieties at the 5′ end and at least two modified moieties at the 3′end.
 115. A molecule according to claim 108, wherein the molecule comprises a nucleotide cap at the 5′ end, the 3′ end or both.
 116. A molecule according to claim 108, wherein the molecule comprises a chemical cap at the 5′ end, the 3′ end, or both.
 117. A molecule according to claim 84, wherein the nuclease is an endonuclease.
 118. A molecule according to claim 1117, wherein the molecule comprises at least one modified moiety between the 5′ and 3′ end.
 119. A molecule according to claim 1117, wherein the molecule comprises a chemical cap between the 5′ end and 3′ end.
 120. A molecule according to claim 84, wherein all of the moieties are nuclease resistant.
 121. A method for inhibiting microRNP activity in a cell, the microRNP comprising a DNA virus microRNA molecule, the DNA virus microRNA molecule comprising a sequences of bases complementary to the sequence of bases in a single stranded anti-DNA virus microRNA molecule, the method comprising introducing into the cell a single-stranded anti-DNA virus microRNA molecule comprising a sequence of a minimum of ten moieties and a maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, each base forming a Watson-Crick base pair with a complementary base, wherein: at least ten contiguous bases of the anti-DNA virus microRNA molecule are complementary to any one of the DNA virus microRNAs shown in Table A2, except that up to thirty percent of the bases may be substituted by wobble base pairs, and up to ten percent of the at least ten moieties are addition, deletions, mismatches, or combinations thereof; and no more than fifty percent of the contiguous moieties contain deoxyribonuleotide backbone units.
 122. An isolated microRNP comprising an isolated nucleic acid molecule according to claim
 69. 